A designed peptide targeting CXCR4 displays anti-acute myelocytic leukemia activity in vitro and in vivo.

Li X, Guo H, Yang Y, Meng J, Liu J, Wang C, Xu H - Sci Rep (2014)

Bottom Line:
We show that E5 has high affinity to multiple AML cells with high CXCR4 level in a concentration dependent manner.E5 can induce concentration-dependent apoptosis in the four AML cell lines tested while did not affect the viability of MS-5 or human umbilical vein cell (ea.hy926) even at 80 µM, both of which have a low level of CXCR4.In vivo experimental results show that immunocompromised mice transplanted with HL-60 cells survived longer when treated with E5 twice a week in comparison to those treated with cyclophosphamide.

ABSTRACTLeukemia cells highly expressing chemokine receptor CXCR4 can actively response to stroma derived factor 1α (CXCL12), trafficking and homing to the marrow microenvironment, which causes poor prognosis and relapse. Here we demonstrate that a novel designed peptide (E5) targeting CXCR4 inhibits CXCL12- and stroma-induced activation in multiple acute myelocytic leukemia (AML) cell lines and displays anti-AML activity. We show that E5 has high affinity to multiple AML cells with high CXCR4 level in a concentration dependent manner. E5 significantly inhibits CXCL12- or murine stromal cell (MS-5)-induced migration of leukemia cells and prevents the cells from adhering to stromal cells. Mechanistic studies demonstrate that E5 down-regulates CXCL12-induced phosphorylation of Akt, Erk, and p38, which affects the cytoskeleton F-actin organization and ultimately results in the inhibition of CXCL12- and stroma-mediated leukemia cell responses. E5 can induce concentration-dependent apoptosis in the four AML cell lines tested while did not affect the viability of MS-5 or human umbilical vein cell (ea.hy926) even at 80 µM, both of which have a low level of CXCR4. In vivo experimental results show that immunocompromised mice transplanted with HL-60 cells survived longer when treated with E5 twice a week in comparison to those treated with cyclophosphamide.

f1: Affinity and kinetic binding of E5 to multiple leukemia cell lines.(a) The CXCR4 level in HL-60, NB4, THP-1 and U937 leukemia cells analyzed by flow cytometry using antibody for CXCR4. (b) Time course of E5 binding amount in the different cells at 10 µM. (c) Affinity of E5 to the different cells at 4 h at 10 µM. Results were obtained from flow cytometry analysis using biotin-labeled E5 and streptavidin-conjugated FITC. (d) and (e) Binding amount of E5 to HL-60 cells after 2 h incubation. The data are presented as mean ± SD (n = 3).

Mentions:
In the first set of experiments, we aimed to determine the affinity of E5 to leukemia cells. Four cell lines including HL-60, NB4, THP-1 and U937 were chosen as cell models because the four cell lines are typical human acute myelocytic leukemia cell lines and highly express CXCR4 in the surface. The CXCR4 level for HL-60, NB4, THP-1 and U937 was detected 98.8%, 99.0%, 99.7% and 94.2% respectively (Fig. 1a). As shown in Figure 1b, the initial binding rate of E5 is associated with the incubation time. The fastest increase in the mean fluorescent intensity (MFI) was observed on NB4, the next two on THP-1 and U937, and the last one on HL-60. The binding platform was reached after 0.5 h incubation and maintained to 4 h in the four cell lines. At the platform stage, the binding amount of E5 to NB4 and THP-1 was similar, while the amount binding to U937 and HL-60 was lower. After 4 h incubation, the percentage of fluorescent cells was 29%, 31.1%, 27.5% and 28.05% for HL-60, NB4, THP-1 and U937 respectively (Fig. 1c). The binding amount of E5 is also concentration dependent (Fig. 1d). As shown in Figure 1e, the MFI of the cells went up gradually when the concentration of E5 was increased from 1 µM to 60 µM, exhibiting a S-shape tendency for three phases, slower increase within 1 µM to 20 µM, faster from 20 µM to 50 µM, and nearly constant from 50 µM to 60 µM.

f1: Affinity and kinetic binding of E5 to multiple leukemia cell lines.(a) The CXCR4 level in HL-60, NB4, THP-1 and U937 leukemia cells analyzed by flow cytometry using antibody for CXCR4. (b) Time course of E5 binding amount in the different cells at 10 µM. (c) Affinity of E5 to the different cells at 4 h at 10 µM. Results were obtained from flow cytometry analysis using biotin-labeled E5 and streptavidin-conjugated FITC. (d) and (e) Binding amount of E5 to HL-60 cells after 2 h incubation. The data are presented as mean ± SD (n = 3).

Mentions:
In the first set of experiments, we aimed to determine the affinity of E5 to leukemia cells. Four cell lines including HL-60, NB4, THP-1 and U937 were chosen as cell models because the four cell lines are typical human acute myelocytic leukemia cell lines and highly express CXCR4 in the surface. The CXCR4 level for HL-60, NB4, THP-1 and U937 was detected 98.8%, 99.0%, 99.7% and 94.2% respectively (Fig. 1a). As shown in Figure 1b, the initial binding rate of E5 is associated with the incubation time. The fastest increase in the mean fluorescent intensity (MFI) was observed on NB4, the next two on THP-1 and U937, and the last one on HL-60. The binding platform was reached after 0.5 h incubation and maintained to 4 h in the four cell lines. At the platform stage, the binding amount of E5 to NB4 and THP-1 was similar, while the amount binding to U937 and HL-60 was lower. After 4 h incubation, the percentage of fluorescent cells was 29%, 31.1%, 27.5% and 28.05% for HL-60, NB4, THP-1 and U937 respectively (Fig. 1c). The binding amount of E5 is also concentration dependent (Fig. 1d). As shown in Figure 1e, the MFI of the cells went up gradually when the concentration of E5 was increased from 1 µM to 60 µM, exhibiting a S-shape tendency for three phases, slower increase within 1 µM to 20 µM, faster from 20 µM to 50 µM, and nearly constant from 50 µM to 60 µM.

Bottom Line:
We show that E5 has high affinity to multiple AML cells with high CXCR4 level in a concentration dependent manner.E5 can induce concentration-dependent apoptosis in the four AML cell lines tested while did not affect the viability of MS-5 or human umbilical vein cell (ea.hy926) even at 80 µM, both of which have a low level of CXCR4.In vivo experimental results show that immunocompromised mice transplanted with HL-60 cells survived longer when treated with E5 twice a week in comparison to those treated with cyclophosphamide.

ABSTRACTLeukemia cells highly expressing chemokine receptor CXCR4 can actively response to stroma derived factor 1α (CXCL12), trafficking and homing to the marrow microenvironment, which causes poor prognosis and relapse. Here we demonstrate that a novel designed peptide (E5) targeting CXCR4 inhibits CXCL12- and stroma-induced activation in multiple acute myelocytic leukemia (AML) cell lines and displays anti-AML activity. We show that E5 has high affinity to multiple AML cells with high CXCR4 level in a concentration dependent manner. E5 significantly inhibits CXCL12- or murine stromal cell (MS-5)-induced migration of leukemia cells and prevents the cells from adhering to stromal cells. Mechanistic studies demonstrate that E5 down-regulates CXCL12-induced phosphorylation of Akt, Erk, and p38, which affects the cytoskeleton F-actin organization and ultimately results in the inhibition of CXCL12- and stroma-mediated leukemia cell responses. E5 can induce concentration-dependent apoptosis in the four AML cell lines tested while did not affect the viability of MS-5 or human umbilical vein cell (ea.hy926) even at 80 µM, both of which have a low level of CXCR4. In vivo experimental results show that immunocompromised mice transplanted with HL-60 cells survived longer when treated with E5 twice a week in comparison to those treated with cyclophosphamide.